Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, provide a unique combination of infinite self-renewal potential and pluripotency, two properties which impart a powerful system for generating normal human somatic cells for developmental studies, toxicity testing, and cellular therapies. Cardiac myocytes are a particularly promising cell type that can be derived from human pluripotent stem cells since cardiac myocytes cannot easily be attained from primary sources or adult stem cells and are of tremendous importance in disease and pharmaceutical evaluation. While substantial progress has been made in generating contractile cardiac myocytes from human pluripotent stem cells, yields remain low and cell populations are heterogeneous, containing atrial, ventricular, and nodal cardiac myocytes, as a result of our lack of fundamental understanding of factors that govern cardiac myocyte development. Current differentiation protocols utilize chemical factors to stimulate cardiogenesis in pluripotent stem cells. In the initial project period we identified cell-cell contact as an important contributor to cardiac myocyte yield from human embryonic stem cells and discovered that an optimum colony size exists for generating cardiac myocytes. This optimum size correlates with reduced signaling through the Want/ -catenin pathway, which has both stimulatory and inhibitory effects on cardiac myocyte development in vivo. In the next project period we propose to elucidate the mechanism by which intercellular interactions affect yield and type specification of cardiac myocytes from pluripotent stem cells. Specifically, we will test the hypothesis that confinement of human pluripotent stem cells in microwells during differentiation affects the yield and type of cardiac myocytes generated by modulating the Want/ -catenin signaling pathway which is regulated by direct cell-cell contact and soluble factors. Our team's expertise in biomaterials development, stem cell biology, signaling pathway analysis, and cardiac myocyte physiology will permit us to construct culture systems that systematically vary colony morphology and presentation of biochemical signaling cues that regulate Want/ -catenin signaling, and quantitatively assess the effects on cardiac myocyte development. Our specific aims to test the hypothesis of this proposal are: 1. quantify the effects of human pluripotent stem cell colony confinement during embryoid body formation and directed differentiation to cardiac myocytes on the yield and phenotype of the resulting cardiac myocytes. 2. Evaluate the mechanistic role of Want/ -catenin signaling in specifying cardiac myocyte differentiation in microwell-confined human pluripotent stem cells. 3. Assess the ability of microwells functionalized with Want signaling regulators to control human pluripotent stem cell differentiation to cardiac myocytes. PUBLIC HEALTH RELEVANCE: Cardiac myocytes derived from human pluripotent stem cells offer a system to study development of the human heart in vitro, a tool to screen the effectiveness of drugs to treat cardiac diseases and to assess cardiac toxicity of other pharmacologic agents, and a potential cellular therapy to treat damaged and diseased hearts. Understanding how cell-cell contact regulates cardiac myocyte differentiation will improve our understanding of human heart development, permit rational design of methodologies to improve yield and functionality of pluripotent stem cell-derived cardiac myocytes, and facilitate translational applications of pluripotent stem cell- derived cardiac myocytes to research and clinical applications.